Glial Cell Line-derived Neurotrophic Factor Gene Research Articles

Adenoviral GDNF gene transfer enhances neurofunctional recovery after recurrent laryngeal nerve injury

To assess the possibility of gene therapy for recurrent laryngeal nerve (RLN) injury, we examined functional and histological recovery after glial cell line-derived neurotrophic factor (GDNF) gene transfer in a rat RLN crush model. Adenoviral vector encoding beta-galactosidase gene (AxCALacZ) or human GDNF gene (AxCAhGDNF) was injected into the crush site of the RLN. Neurons in the nucleus ambiguus on the crushed side were labeled with X-gal or GDNF immnohistochemistry after AxCALacZ or AxCAhGDNF injection. Reverse transcription-polymerase chain reaction analysis revealed expression of human GDNF mRNA transcripts in brainstem tissue containing the nucleus ambiguus on the crushed side after AxCAhGDNF injection. Animals injected with AxCAhGDNF displayed significantly improved motor nerve conduction velocity of the RLN and recovery rate of vocal fold movement at 2 and 4 weeks after treatment as compared to controls. AxCAhGDNF-injected animals showed a significantly larger axonal diameter and improved remyelination in crushed RLN as compared to controls. Adenoviral GDNF gene transfer may thus promote laryngeal function recovery after RLN injury. Inoculation of adenoviral vector containing the GDNF gene at the site of damage soon after nerve injury may assist patients with laryngeal paralysis caused by nerve injury during head and neck surgery.

Gene therapy using adenovirus

Glial cell line-derived neurotrophic factor (GDNF) has a strong neuroprotective property. It prevents neuronal cell death in various situations, and intracerebroventricular injection of this molecule actually reduced ischemic brain injury in vivo. However, GDNF needs to be given intracerebroventricularly because the blood-brain barrier inhibits its entry into the brain parenchyma from intravascular space. In order to get over this problem, gene transfer could be a promising method. Adenovirus is an appropriate vector to transfer genes into the brain, because this virus successfully infects non-dividing cells as neurons. In the ischemic brain, however, initiation of protein synthesis is inhibited, and thus, whether transferred gene becomes translated into protein remained uncertain. In an attempt to clarify this issue, we injected adenovirus containing lacZ gene into the brain after transient or permanent middle cerebral artery (MCA) occlusion. In the brain after transient MCA occlusion, lacZ activity was not or only minimally observed in the reperfused brain until 2 days. However, the activity dramatically increased at 7 days of reperfusion at a level similar to that of the control, which diminished by 21 days. In the brain with permanent MCA occlusion, lacZ activity was increased from 8 h to 2 days in the MCA territory, which was less stronger than that of the control. Based on the finding of the successful expression of transferred lacZ gene, we injected adenovirus containing GDNF gene into the brain followed by making transient MCA occlusion. The infarction volume was measured with 2,3,5-triphenyltetrazolium chloride staining method, and it was revealed that GDNF-containing adenovirus reduced the infarction volume at 1 day of reperfusion. The numbers of TUNEL, immunoreactive caspase-3, and cytochrome c-positive neurons were markedly reduced in the GDNF gene transferred group, suggesting that amelioration of brain damage was caused by reduction of apoptotic signals activation. As the next step, we investigated therapeutic time window of GDNF gene-containing adenovirus. In the group of adenovirus injection at just before or just after the MCA occlusion, the infarction volume was significantly smaller than the control group. On the other hand, adnenovirus injection at 1 h after the reperfusion failed to reduce the infarction volume. This means that the GDNF gene transfer with use of adenovirus could be effective for treating stroke, if the vector was applied at an early time after the ischemic insult.

Hypothalamic rAAV-mediated GDNF gene delivery ameliorates age-related obesity

Intraventricular delivery of glial cell line-derived neurotrophic factor (GDNF) results in weight loss. We hypothesized that this effect of GDNF was likely mediated via its effects on dopaminergic neurons in the hypothalamus. Continuous rAAV-mediated GDNF expression in the hypothalamus of young and senescent rats resulted in weight loss compared to controls. However, GDNF-induced weight loss was unrelated to alterations in hypothalamic dopamine levels. The weight loss was associated with decreased food intake and increased energy expenditure, but these effects were not mediated by changes in hypothalamic NPY or POMC expression. Moreover, uncoupling protein 1 levels were unchanged in brown adipose tissue (BAT). The reduction in weight and adiposity were as great or greater in the aged rats even though aged rats are generally resistant to weight loss therapies. In summary, central GDNF gene delivery reduces weight and adiposity in young and aged rats through decreased food intake and increased energy expenditure. Our observations in aged rats suggest that GDNF may be especially effective in reducing obesity in aged obese rats.

549. Peripheral Gene Delivery of Glial Cell Line-Derived Neurotrophic Factor Alleviated Neurological Deficits in Diabetic Rats

The neurological deficit in peripheral nerves is one of the major complications associated with diabetes. Deprivation of trophic factors may contribute to the pathogenesis of diabetic neuropathy while restored expression of neurotrophic factors could ameliorate such sensory abnormalities. Glial cell line-derived neurotrophic factor (GDNF) is a potent neurotrophic factor that effectively prevents neuronal death and restores the synapse conduction during trauma of central nervous system. In the present study, we evaluated the therapeutic potential of GDNF gene delivery for neuropathy in diabetic rats. After injection of streptozotocin for 14 days, a reduction in nerve conduction velocity (NCV) with a concomitant increment in H-reflex was detected in diabetic rats by electrophysiological studies. Single, intramuscular (IM) injection of adenovirus encoding GDNF (Ad-GDNF) led to systemic GDNF expression for at least 35 days. Besides, the declined GDNF level in sciatic nerve was also restored by GDNF gene delivery. After gene delivery for 2-4 weeks, Ad-GDNF-treated rats exhibited a significant increment in NCV (P<0.05) and a concomitant decrease in H-reflex latency (P<0.05) comparing with animals of control groups. IM GDNF gene delivery was well tolerant to diabetic rats without affecting their biochemical parameters Therefore, peripheral GDNF gene delivery may hold potential for treatment of diabetic neuropathy.

Lentiviral gene delivery of GDNF into the striatum of R6/2 Huntington mice fails to attenuate behavioral and neuropathological changes

Transgenic R6/2 mice, which express exon 1 of the human mutant Huntington disease gene, develop behavioral and neuropathological changes that bear some resemblance to the human disease. Several studies have shown that elevated glial cell line-derived neurotrophic factor (GDNF) levels can exert neuroprotective effects in animal models of Huntington disease that are based on intrastriatal injections of excitotoxins. Therefore, the aim of the present study was to examine whether intrastriatal delivery of the GDNF gene by lentivirus (LV-GDNF) could provide structural and functional protection in R6/2 transgenic mice. Four- to 5-week-old mice were left untreated or alternatively received intrastriatal injections of either LV-GDNF or the same viral vector encoding green fluorescent protein (GFP) (LV-GFP) as a control. During the 4-week follow-up period, there was the expected deterioration in performance of the R6/2 mice in paw clasping, rotarod, and open field tests, and the LV-GDNF treated mice showed no improvement over controls. ELISA showed that the LV-GDNF-injected animals had a significant increase in GDNF level in the striatum, and immunohistochemical analysis revealed that GDNF was also overexpressed in brain regions receiving striatal projections. However, GDNF overexpression had no effect on the neuropathological changes examined. Thus, there were no significant differences in the number of EM-48-positive intraneuronal huntingtin inclusions, number of BrdU-positive cells and size of striatal neuronal cross-sectional area. These results suggest that intrastriatal lentiviral vector transfer of GDNF, performed at 5 weeks of age, does not ameliorate neurological and behavioral impairments in the R6/2 transgenic mice model of HD. Further studies are, however, needed to investigate if GDNF given at earlier time points is beneficial.

Intrathecal gene delivery of glial cell line-derived neurotrophic factor ameliorated paraplegia in rats after spinal ischemia

Paraplegia is a catastrophic complication of thoracic aortic surgery. At present, there is no effective mean to prevent the ischemia-induced spinal cord trauma. Gene delivery of neurotrophic factors may hold promises for prevention of spinal injury. In the present study, we evaluated the effect of glial cell line-derived neurotrophic factor (GDNF) gene delivery on prevention of the pathological changes due to spinal ischemia. Recombinant adenovirus vectors encoding GDNF (Ad-GDNF) and green fluorescent protein (Ad-GFP) were used for gene transfer studies. Treatment with cobalt chloride induced dose-dependent bcl-2 and synaptophysin downregulation in spinal neuronal cells, which could be effectively reversed by GDNF gene transfer. Intrathecal injection of Ad-GDNF led to maximal GDNF expression in spinal cord within 2–7 days. Thus, after intrathecal administration of adenovirus vectors for 3 days, Sprague–Dawley rats received transient aortic occlusion to induce spinal ischemia and were monitored for behavior deficits. The Ad-GDNF-treated rats showed significantly lower paraplegia rate (40%) than that of Ad-GFP- or saline-treated groups (75–85%; P < 0.01). In addition, the Ad-GDNF-treated rats exhibited significantly improved locomotor function comparing with rats of control groups ( P < 0.001). Histological analysis revealed that GDNF gene delivery profoundly attenuated the infiltration of leukocytes in spinal cord after ischemic insults. Furthermore, GDNF gene delivery prominently attenuated the ischemia-induced neuronal loss in dorsal horn lamina VI–VIII and reduction in synaptophysin expression in spinal cords. In conclusion, GDNF gene transfer confers protection to the neuronal cells and synapses networks, thereby alleviated the paraplegia due to spinal ischemia.

Long-Term Safety of GDNF Gene Delivery in the Retina

Purpose: To examine retinal function after the long-term, gene therapy–delivered expression of exogenous glial cell line–derived neurotrophic factor (GDNF). Methods: Forty Sprague-Dawley rats each received an intravitreal injection of recombinant adeno-associated virus expressing GDNF (rAAV-GDNF) in their right eye. The left eye was untreated. One year after viral transduction in ocular tissues, retinal morphology and function were compared between rAAV-GDNF–injected and normal naïve eyes. Synthesis and accumulation of GDNF within the retina were immunohistologically confirmed using enzyme-linked immunosorbent assay. Morphological analyses included light microscope examination of retinal sections and the counting of retinal ganglion cells. Inflammation by infiltration of leukocytes in retina was assessed immunohistochemically. Retinal function was assessed using electroretinography. Results: GDNF expression was confirmed. There was no obvious abnormality in retinal section or increased infiltration by leukocytes after retinal transduction of rAAV-GDNF for 1 year. Counts of retinal ganglion cells were not decreased in rAAV-GDNF–injected eyes. There were no statistical differences in amplitude as well as latency of the electroretinogram-determined a- and b-waves between transduced and untreated eyes. Conclusions: Long-term expression of GDNF within the eyes can be achieved by intravitreal injection of rAAV vectors in the absence of morphological or functional deficits in the retina.

Effect of GDNF gene transfer into axotomized retinal ganglion cells using in vivo electroporation with a contact lens-type electrode

We developed an in vivo electroporation method to introduce foreign genes into retinal ganglion cells (RGCs). After the intravitreous injection of the plasmid gene (20 mug), five electric pulses (6 V/cm, 100 ms duration) were each delivered twice with 5 min interval to the rat eye using a contact lens-type electrode (cathodal) attached to the cornea and a needle electrode (anodal) inserted to the middle of the forehead. The efficiency of the genetic introduction into RGCs and tissue damage to the eyeball was evaluated using a green fluorescent protein (GFP) gene, TUNEL and histological observation. DiI retrograde labeling revealed that 24.4 +/- 4.7% of all RGCs were electrointroduced with the GFP gene. TUNEL and histological analysis showed a few tissue damages in the cornea, lens and retina. To confirm whether this method can actually rescue damaged RGCs, glial cell line-derived neurotrophic factor (GDNF) was electrointroduced into RGCs after optic nerve transection. After the electrointroduction, a significant increase in the number of surviving RGCs was observed 2 and 4 weeks after the optic nerve transection, and the decrease of caspase 3 and 9 was detected by RT-PCR. These results suggest that this method may be useful for the delivery of genes into RGCs with simplicity and minimal tissue damage.

Motoneurons crave glial cell line-derived neurotrophic factor

This is a commentary on the developmental and therapeutic relevance of recent studies in the glial fibrillary acid protein (GFAP)-glial cell line-derived neurotrophic factor (GDNF) transgenic mouse reported by Zhao et al. (2004). This interesting study demonstrated that increased expression of GDNF in astrocytes increases the number of neighboring motoneurons of certain motoneuron subpopulations by diminishing programmed cell death during development. In addition, astrocyte-derived GDNF was shown to protect facial motoneurons from injury-induced cell death. Since this is the first direct demonstration that secretion of GDNF from astrocytes in the CNS can affect motoneuron development in utero and motoneuron survival after axotomy, novel approaches for motor neuron disease are suggested. The known target neurons that respond to GDNF are reviewed, as are studies using GDNF gene delivery in animal models of amyotrophic lateral sclerosis (ALS). It is postulated that GDNF is a factor to which many motoneurons respond along their whole extent from soma to axon to terminal.

Effects of hyperbaric oxygen on GDNF expression and apoptosis in spinal cord injury

The effects of hyperbaric oxygen treatment on the progress of secondary damage following traumatic spinal cord injury were investigated. The early onset of hyperbaric oxygen treatment significantly diminished the number of apoptotic cells 1 day after the injury. However, hyperbaric oxygen did not influence the proliferation of macrophages or activated microglia. The gene expression of glial cell line-derived neurotrophic factor (GDNF) and inducible nitric oxide synthetase (iNOS) was significantly attenuated 1 day after the injury in the hyperbaric oxygen groups compared with the control group. The down-regulation was confirmed by immunohistochemical staining. Early hyperbaric oxygen treatment was shown to effectively suppress the progress of apoptosis perhaps via the inhibition of iNOS gene despite the down-regulation of the GDNF gene.

Suppression of GDNF production by MPSS treatment following spinal cord injury in the rat

The synthetic glucocorticosteroid methylprednisolone sodium succinate (MPSS) has been clinically used for the treatment of acute spinal cord injury (SCI) to promote the recovery of neurological functions. However, the mechanisms of its beneficial actions are not entirely understood. Experimental evidence suggests that MPSS may contribute to some extent to neuroprotection in SCI. On the other hand, glial cell line-derived neurotrophic factor (GDNF) acts as a potent survival factor for several neuronal populations, providing a therapeutic promise for neurological disorders. This experiment demonstrated, for the first time, the time-effect relationship of MPSS on GDNF gene expression and protein synthesis in the injured spinal cord tissues.

Hydrophobic dipeptide Leu-Ile protects against neuronal death by inducing brain-derived neurotrophic factor and glial cell line-derived neurotrophic factor synthesis.

We investigated whether certain hydrophobic dipeptides, Leu-Ile, Leu-Pro, and Pro-Ile, which partially resemble the site on FK506 that binds to immunophilin, could stimulate glial cell line-derived neurotrophic factor (GDNF) and brain-derived neurotrophic factor (BDNF) synthesis in cultured neurons and found only Leu-Ile to be an active dipeptide. Leu-Ile protected against the death of mesencephalic neurons from wild-type mice but not from mice lacking the BDNF or GDNF gene. Next, we examined the effects of i.p. or i.c.v. administration of Leu-Ile on BDNF and GDNF contents. Both types of administration increased the contents of BDNF and GDNF in the striatum of mice. Also, peripheral administration of Leu-Ile inhibited dopaminergic (DA) denervation caused by unilateral injection of 6-hydroxydopamine (6-OHDA) into the striatum of mice. The number of rotations following a methamphetamine challenge was lower in the Leu-Ile-treated group than in the nontreated group. Next, we compared the calcineurin activity and immunosuppressant activity of Leu-Ile with those of FK506. Leu-Ile was not inhibitory toward calcineurin cellular activity in cultured neuronal cells. Furthermore, Leu-Ile did not suppress concanavalin A (ConA)-induced synthesis/secretion of interleukin-2 by cultured spleen cells, suggesting that the immunosuppressant activity of Leu-Ile may be negligible when used as a therapeutic tool for neurodegenerative diseases.

Delayed gene therapy of glial cell line-derived neurotrophic factor is efficacious in a rat model of Parkinson's disease

Gene transfer of glial cell line-derived neurotrophic factor (GDNF) in rodent models of Parkinson's disease (PD) has been shown to protect against neurodegeneration either prior to or immediately after neurotoxin-induced lesions; however, the nigrostriatal pathway was largely intact when gene delivery was completed in these models, which may not accurately reflect the clinical situation encountered with Parkinson's patients. In this study, replication-incompetent adenoviral vectors encoding the rat GDNF gene were administered into the striatum 4 weeks following 6-hydroxydopamine (6-OHDA) injection in the unilateral striatum, more closely resembling fully developed PD. Apomorphine-induced rotational behavior testing was performed every week following 6-OHDA injection. At the 10th week after gene transfer, the striatal dopamine concentrations were measured by HPLC with an electrochemical detector and the number of tyrosine hydroxylase (TH)-positive dopamine neurons in the substantia nigra (SN) was determined by immunohistochemistry. Injection of 6-OHDA into the striatum produced stable increases in rotation, which reached a plateau between 4 and 5 weeks post-injection. The number of TH-positive neuron in the SN and dopamine levels in the striatum was significantly lower in the 6-OHDA group compared to the normal group. Gene transfer of GDNF, but not β-galactosidase, significantly increased the number of TH-positive neurons and dopamine levels, with a subsequent behavioral recovery between 5 and 10 weeks following GDNF transduction. These findings demonstrate that adenovirus-mediated gene transfer of GDNF is efficacious even in the late stages of 6-OHDA-induced PD rats. They also provide further evidence on the effectiveness of GDNF-based gene therapy for experimental Parkinson's disease.

Neurofunctional recovery after GDNF gene transfer in the recurrent laryngeal nerve-injured rat

Problem: To assess the possibility of gene therapy for recurrent laryngeal nerve (RLN) injury, we examined the neuroprotective effect after glial cell line-derived neurotrophic factor (GDNF) gene transfer into nucleus ambiguus in a rat vagal nerve avulsion model. Morphological preservation of motoneurons has previously been demonstrated (Brain Res 962: 61–67, 2003). However, functional recovery of RLN after gene therapy has not been demonstrated. In the present study, we examined neurofunctional recovery after GDNF gene transfer in a rat RLN injury model. Methods: The left RLN of Sprague-Dawley rats was crushed for 60 seconds with forceps. Immediately after the crush and confirmation of left fixed vocal fold, an adenoviral vector encoding beta-galactosidase gene (AxCALacZ) or GDNF (AxCAhGDNF) was directly injected into the crushed site of RLN. Transgene expression by AxCALacZ in nucleus ambiguus was examined by X-gal histochemistry. Measurement of conduction velocity (CV) of RLN and laryngoscopy to check motion recovery of the left vocal fold were performed 2 and 4 weeks postoperation. The cross-section of RLN was histologically examined after epon/toluidine blue staining. Results: Motoneurons and their neurites in nucleus ambiguus were labeled with X-gal staining 4 days after AxCALacZ injection, indicating successful induction of foreign gene into the nucleus ambiguus by retrogradely transported adenoviral vector from the crushed site. In comparison between AxCAhGDNF-injected, AxCALacZ-injected, and noninjected animals, significantly better CV of RLN and better vocal fold motion recovery rate were observed in AxCAhGDNF-injected animals 2 and 4 weeks postoperation. RLN cross section of AxCAhGDNF-injected animals showed better myelination compared to the controls. Conclusion: Injection of adenoviral vectors into the crushed site of RLN successfully induced the foreign gene into motoneurons in nucleus ambiguus. Adenoviral GDNF gene transfer after RLN crush improved the CV of RLN and motion recovery of the paralyzed vocal fold. Significance: Adenoviral GDNF gene therapy may provide neurofunctional recovery in humans with a paralyzed vocal fold. Support: None reported.

3′ UTR (AGG) n repeat of glial cell line-derived neurotrophic factor (GDNF) gene polymorphism in schizophrenia

Association studies on gene polymorphisms of neurotransmitter systems have hypothesized an involvement of dopamine receptors in susceptibility to schizophrenia. However, structural and morphological abnormalities in different brain regions of schizophrenic patients support neurodevelopmental etiology for schizophrenia and neurotrophic factor genes could be candidates for genetic studies. The glial cell line-derived neurotrophic factor (GDNF) is a neurotrophic and potential differentiation factor for dopaminergic systems. We have performed, in an Italian sample, an association study on 3′ UTR (AGG) n repeat in GDNF gene. Our results have evidenced a difference in the allele frequencies between patients and controls (CLUMP (T1) χ 2=17.365, df=9, P=0.043) and the (AGG) n ≥15 alleles (Fisher Exact Test (two side) χ 2=11.818, df=1, P=0.0003) were more present in the controls group. Similarity, the carriers of (AGG) n ≥15 (OR=0.176 95% CI: 0.060–0.520) were more present in the same group. These results support that the (AGG) n ≥15 alleles could be protective factors against schizophrenia and thus they suggest a possible involvement of GDNF gene in the genetic liability to illness.

Olfactory ensheathing cells genetically modified to secrete GDNF to promote spinal cord repair

Olfactory ensheathing cell (OEC) transplantation has emerged as a very promising therapy for spinal cord repair. In this study, we tested the ability of genetically modified OECs to secrete high levels of glial cell line-derived neurotrophic factor (GDNF) to promote spinal cord repair. The GDNF gene was transduced into OECs using a retroviral-based system. The engineered OECs were first characterized by their ability to express and secrete biologically active GDNF in vitro. After implantation into the spinal cord of adult rats with complete spinal cord transection, OEC survival and GDNF production were examined. The locomotor functions of animals were assessed and axon regeneration was evaluated at the morphological level. To our knowledge, we report for the first time that the genetically modified OECs are capable of producing GDNF in vivo to significantly improve recovery after spinal cord injury (SCI). This work combined the outgrowth-promoting property of OECs with the neuroprotective effects of the additionally overexpressed neurotrophic factors and opens new avenues for the treatment of SCI.

Real-time quantitative RT-PCR for low-abundance transcripts in the inner ear: analysis of neurotrophic factor expression

Real-time quantitative reverse transcription-PCR is a highly sensitive technology that allows high throughput quantification of gene expression. Application of this technique to the inner ear is potentially very important, but is not straightforward because tissue harvesting can be challenging, RNA yield from individual inner ears is low, and cDNA synthesis from scant RNA can be inefficient. To overcome these challenges, we tested many parameters and reagents, and developed an approach to reliably quantitate small changes in low-abundance transcripts. Using this technique we demonstrate the presence and quantify amounts of the neurotrophic factors neurotrophin 3 (NT-3), brain-derived neurotrophic factor (BDNF) and glial cell-line-derived neurotrophic factor (GDNF), in the cochlea and vestibular end organs of postnatal murine inner ear (P26). We show that out of the factors tested, BDNF is the only one differentially expressed between the cochlea and vestibular end organs, being 23.4±0.3 times more abundant in the vestibular end organs. Within the cochlea, GDNF gene expression is 4.9±0.2 times greater than NT-3 expression. Within the combined vestibular end organs, BDNF expression is 43.0±1.5 times greater than NT-3 expression. Our results suggest that neurotrophic factors continue to play a role in the postnatal inner ear, in addition to their previously shown essential role during development.

Protective effects of intracerebral adenoviral-mediated GDNF gene transfer in a rat model of Parkinson's disease

This study focuses on the potential protective effects of intracerebral adeno-viral mediated glial cell line derived neurotrophic factor (GDNF) gene transfer in a rat model of Parkinson's disease (PD). Thirty-five SD rats were divided into three groups to receive perinigral injections of recombinant adenovirus encoding GDNF (Ad-GDNF), LacZ (Ad-LacZ) or PBS, respectively. One week later, an intrastriatal injection of 6-hydroxydopamine (6-OHDA) was administered to induce the progressive degeneration of dopaminergic neurons. Immunohistochemistry showed that GDNF treatment prior to neuronal damage could promote survival and morphological recovery of tyrosine hydroxylase (TH)-positive neurons in the midbrain. Approximately 70% of nigral TH-positive cells survived in the Ad-GDNF group, compared to approximately 30% for the Ad-LacZ or PBS control group. Histochemical analysis of monoamine levels in the striatum demonstrated that the dopamine content was higher for the Ad-GDNF group than the control groups. Similarly, Ad-GDNF treated animals showed improved apomorphine-induced rotational behavior. The exogenous GDNF gene was efficiently expressed in the brain as detected by ELISA. This work demonstrates that intracerebral adeno-viral mediated GDNF gene transfer can protect dopaminergic neurons in vivo from 6-OHDA-induced injuries. The approach used in this study could potentially be used therapeutically in patients with PD and further work is required to explore this idea in depth.

Gene transfer of glial cell line-derived neurotrophic factor promotes functional recovery following spinal cord contusion

Neuronal cell death and the failure of axonal regeneration cause a permanent functional deficit following spinal cord injury (SCI). Administration of recombinant glial cell line-derived neurotrophic factor (GDNF) has previously been reported to rescue neurons following severe SCI, resulting in improved hindlimb locomotion in rats. In this study, thus, GDNF gene therapy using an adenoviral vector (rAd-GDNF) was examined in rats following SCI induced by dropping the NYU weight-drop impactor from a height of 25 mm onto spinal segment T9–T10. To evaluate the efficacy of intraspinal injection of recombinant adenovirus into the injured spinal cord, we observed green fluorescent protein (GFP) gene transfer in the contused spinal cord. GFP was effectively expressed in the injured spinal cord, and the most prominently transduced cells were astrocytes. The expression of GDNF was detected only in rats receiving rAd-GDNF, not the controls, and remained detectable around the injured site for at least 8 days. Open-field locomotion analysis revealed that rats receiving rAd-GDNF exhibited improved locomotor function and hindlimb weight support compared to the control groups. Immunohistochemical examination for the neuronal marker, calcitonin gene-related peptide (CGRP), showed an increase in CGRP+ neuronal fibers in the injured spinal cord in rats receiving rAd-GDNF treatment. Collectively, the results suggest that adenoviral gene transfer of GDNF can preserve neuronal fibers and promote hindlimb locomotor recovery from spinal cord contusion. This research should provide information for developing a clinical strategy for GDNF gene therapy.

Therapeutic time window of Ad-GDNF after transient middle cerebral artery occlusion in rat

Time-dependent influence of adenovirus-mediated glial cell line-derived neurotrophic factor (GDNF) gene (Ad-GDNF) was examined after 90 min of transient middle cerebral artery occlusion (MCAO) in rats. Treatment with Ad-GDNF significantly reduced the infarct volume when immediately administered after reperfusion, but became insignificant when administered at 1 h after the reperfusion as were the cases treated with vehicle and adenoviral vector containing the Escherichia coli lacZ gene (Ad-LacZ)-treated groups. The protective effect of GDNF was related to the significant reduction of the number of terminal deoxynucleotidyl transferase-mediated dUTP-biotin in situ nick end labeling (TUNEL)-positive cells as well as immunohistochemical positive cells for active caspase-3 but not caspase-9. These results showed that exogenous GDNF gene transfer successfully reduced the infarct size in a time-dependant manner by suppressing active caspase-3 but not active caspase-9. However, the therapeutic time window was shorter than the effect of GDNF protein itself previously reported.